New insights about Na+/Ca2+ exchangers and protein glycosylation in human cells

Abstract

Na+/Ca2+ exchangers (NCX and NCKX proteins) contribute to Ca2+ homeostasis and recent studies have demonstrated the expression of NCX1 (SLC8) and NCKX1 (SLC24) proteins in human platelets. A tight ([Ca2+]i) is necessary for platelets to prevent inappropriate thrombus formation or bleeding due to altered platelet aggregation, a severe clinical manifestation in congenital disorders of glycosylation (CDG) PMM2-CDG patients. Very little is known about glycosylation and Ca2+ uptake. In this study, we propose to examine Na+/Ca2+ activity (45-Ca2+ uptake) and protein glycosylation in human cells. Immunopurified NCX1 and NCKX1 proteins from microsomal fractions of human platelets were detected with anti-SLC8 antibody and anti-SLC24 antibody, respectively, and lectin staining (concanavalin A (Con A) and wheat germ agglutinin (WGA)). Additionally, enzymatic N- and O-deglycosylation strategies (PNGase F and O-glycosidase digestion) were assayed. In healthy control subjects, we observed N-linked glycans attached to NCX1 and NCKX1 proteins and O-linked sialo oligosaccharides attached to NCKX1. To better understand the clinical relevance of altered protein N-glycosylation, we analyzed the 45-Ca2+ uptake in PMM2-CDG platelets and transfected NCX1 cDNA in tunicamycin-treated HEK293 cells. Western blot showed that 45-Ca2+ uptake and NCX1 protein were greatly diminished. We present evidence that suggests for the first time that N-hypoglycosylation alters Na+/Ca2+ exchange in platelets from CDG patients or tunicamycin-treated HEK293 cells. Additional studies will be necessary to further elucidate the structure of glycans bound to these proteins and the pathological aspects of cell hypoglycosylation.